Project Summary Antibiotic resistance has recently emerged as a global threat for healthcare systems. An increasing number of pathogenic bacteria are acquiring antibiotic resistance, and new forms of resistance are continuously emerging with alarming speed across international boundaries.1 In the U. S. alone, the Center for Disease Control and Prevention (CDC) has estimated 2 million patients per year are directly affected by antibiotic-resistant pathogens, leading to more than 23,000 deaths.2 Providing rapid, definitive and cost effective antibiotic susceptibility testing (AST) will be of increasingly vital importance in controlling this burgeoning problem. While legacy methods generally require overnight culturing, the time-urgency of determining effective antibiotics has prompted a push for rapid AST that can perform in a few hours. Unfortunately, some of the newest rapid AST instruments are either not suited to multiplexing, or are an order of magnitude more costly than legacy methods, discouraging their widespread use. A rapid multiplexed AST platform that provides minimum inhibitory concentrations (MICs) within hours at comparable cost to current methods will enable rapid determination of effective targeted therapy, resulting in short hospital stays and fewer additional laboratory tests, and decreasing morbidity and mortality along with associated healthcare expenses. Specific Technologies has introduced and with NIAID support commercially developed a culture system that combines detection with organism ID.3, 4 Using a printed array of chemically responsive colorimetric indicators introduced into culture headspace, the species-specific pattern of volatiles emitted during growth produce a species-specific pattern, which we have shown to reduce detection time and enable Gram status and species ID to be determined hands-free during culture. Here, we present data showing that the use of this system can be extended to very rapidly, order 1 hour, ascertain phenotypic antibiotic susceptibility. The preliminary data demonstrate that introduction of antibiotic to positive blood culture prompts a susceptibility-dependent and antibiotic concentration-dependent shift in volatile sensor response within <60 minutes. Recognizing that a commercial AST instrument must be multi-well, we have performed pilot study in which the positive sample is removed from a blood culture bottle and divided into a multi-chamber setting, in which even at pilot study stage we demonstrate a < 2 hrs phenotypic AST, measuring values matching the results of the CLIA-compliant Sensititre gold standard. Moreover, the disposable is inherently low cost, as it only depends upon an inexpensive printed sheet of CSA?s. Finally, not only is this method intrinsically low-cost but low-skill compatible, as there is no sample preparation from positive blood culture. It is thus very well-suited for both 24-hour/day developed world labs, where it will speed time-to-answer, as well as the low-skill environments of the developing world. In this direct-to-Phase II application, we propose to use our CSA technology to systematically quantify, and validate the parameter-dependence of this system and then engineer and produce a benchtop instrument suitable for preclinical and clinical testing, which will provide a low cost, highly automated multiplexed phenotypic susceptibility determination directly from a positive blood sample in less than 90 minutes. We propose to develop and commercialize an instrument that integrates our rapid AST capability with our Spec80 blood culture and species ID system that is being commercialized now, offering a uniquely streamlined and low cost system with by far the fastest yet proposed time from patient sample to detection, ID and phenotypic multiplexed quantitative AST.